The Functional Mechanism Of Hydroxycholesterol Inmigration And Invasion Of Lung Adenocarcinoma

Background and ObjectiveLung cancer is a leading cause of cancer-related death worldwide.Lung adenocarcinoma(ADC)is the main common subtype of lung cancer.Metastasis and invasion have been great challenges in lung ADC therapy.However,the molecular mechanisms that underlie invasion and metastasis are complex and involve many molecules and pathways.Liver X receptors(LXRa/NRlH3 and LXRβ/NR1H2)are nuclear receptors that function as ligand-activated transcription factors regulating lipid metabolism and inflammation.LXR could be activated by natural ligands:oxysterols(like 25-hydroxycholesterol and 27-hydroxycholesterol),as well as synthetic agonists such as T0901317(with an ECso-50nM).25-hydroxycholesterol(25-HC)and 27-hydroxycholesterol(27-HC)are two types of oxysterol that enzymatically produced by cholesterol hydorxylase in various organs and severed as ligands of LXR.25-HC is a potent regulator of LXR-mediated pathways,that impact on brain lipid homeostasis,inflammation and the immune response.Beside,25-HC can further inhibit cancer cell proliferation including glioblastoma,breast and prostate cancer cells as well as prostate cancer xenografts.Previous research revealed that 27-HC exhibited significant ER and LXR partial agonist activity at concentrations expected to be found in humans.Additionally,27-HC notably promotes the metastasis of breast cancer by activating this receptor,but LXR activation suppresses the metastasis of melanoma.These findings prompted us to evaluate the extent to which hydroxy cholesterol(HC)affects tumor pathophysiology and further invasion.This study was to investigate whether 25-HC or 27-HC can influence the invasion and metastasis of ADC cells and research its mechanism to provide new evidence for the treatment of tumor.MethodsIn this study,we created monoculture and coculture systems with ADC cells A549 or NCL-H1975(apical side)and human macrophage THP1 cells(basolateral side).To exclude the effects of hydroxycholesterols on cell viabilities,CCK-8 method was used to evaluate the effect of HC on cells proliferation and select appropriate concentration for scratch wound and invasion assay.We chose 0.1 μ1 of 25-HC and 1 μM of 27-HC for the following experiments.For migration assay,the ADC cells were seeded in 6-well microplates and cultured for overnight.A linear wound was subsequently generated in the monolayer using a sterile 200 uL plastic pipette tip to produce an approximately 1 mm wide scratch.In monoculture system,the medium was added different drugs as shown above or knockdown the gene of LXR.In coculture system,the supernatant was collected form THP1-derived macrophages which had been treated with above drugs for 24 hours.Photos were captured in three defined fields at 0 hour and 24 hours,respectively.For invasion assay,the ADC cells were seeded into upper chambers with serum free medium,the complete media contains above drugs were added to bottom chambers.The supernatant harvested from the monoculture or coculture system was used to measure the levels of cytokines(IL-1β and TGF-β3)using enzyme linkedimmunosorbent assay(ELISA),according to manufacturer’s protocol.Western blot analysis was performed to detected the expression of LXR and Snail.Results1.25-HC has no significant effect on cell proliferation at 0.1 μM,but it can inhibit cell proliferation at the concentration of 1 μM up to 25μM in the two lung adenocarcinoma cell lines.For 27-HC,there are no effect on cell proliferation at theconcentration of 0.1 μM and 1μM,but it promotes cell proliferation at the concentration of 10μM up to 50p.M.T0901317 shows no effect on the proliferation of two ADC cells.2.In monoculture system,hydroxycholesterol(0.1 μM for 25-HC;1μM for 27-HC)can slightly promote migration and invasion compared to the control.In cocultuer system,the migration and invasion were significantly accelerated in both lung adenocarcinoma cell lines.Additionally,the LXR agonists T0901317 had the similar effect to hydroxycholesterol.3.The ELISA assays showed that exposure of the THP1-derived macrophages to hydroxycholesterols reduced the secretion of IL-1β and TGF-β1 compared to the control group.Intriguingly,coculture of the THP1-derived macrophages with the ADC cells increased the levels of IL-1β,which further enhanced by treated with hydroxycholesterols,which was different from monoculture.4.Hydroxycholesterols treatment increased the expression of Snail and LXR in the monoculture system,which was further enhanced in the coculture system.5.IL-1β treatment stimulated ADC cell migration and invasion,andincreased Snail expression in both the monoculture system and in the coculture system,but had no significant effect on LXR expression.6.LXR knockdown remarkably suppressed the 25-HC-induced Snail expression in both the monoculture and coculture systems,but did not affect the IL-1β-induced Snail expression.7.LXR knockdown inhibited the 25-HC-induced migration and invasion of the ADC cells in both the monoculture and coculture systems,but did not affect IL-1β-induced migration and invasion of ADC cells.ConclusionIn this study,we demonstrated that 25-HC did not affect ADC cell proliferation at the concentration of 0.1μM.With the increase of concentration,25HC could restrain cell proliferation.In addition,25HC could promote migration and invasion without affecting cell proliferation,especially after coculture with THP1-derived macrophages.At the same time,the expression of Snail and LXR,the receptor of 25HC,are also up-regulated.LXR knockdown inhibited the 25-HC-induced migration and invasion of the ADC cells in both culture systems as well as the expression of Snail.Taken together,25HC can promote migration and invasion of ADC cells by stimulating LXR.We also found that 25HC significantly stimulated IL-1β secretion in a coculture system and increased the expression of Snail.Further investigation shown that IL-1βtreatment mimicked the effect of 25-HC on Snail expression as well as ADC cell migration and invasion.LXR knockdown blocked the 25-HC-induced Snail expression in both ADC cell monocultures and in cocultures of ADC cells and macrophages.However,LXR knockdown did not affect the IL-1β-induced events,including Snail expression and ADC cell migration and invasion.Our results indicated that 25-HC promoted LXR-dependent migration and invasion in ADC cell monocultures,and the 25-HC-induced IL-1β secretion in the coculture system could accelerate the ADC cell migration and invasion in an LXR-independent manner.When the concentration weas lower than 1 μM,27HC had no effect on ADC cell proliferation.As the concentration increased,27HC promoted cell proliferation.T0901317,the agonist of LXR,has no effect on cells proliferation at 50nM to 10μM.Additionally,27HC and T0901317 could promote the migration and invasion,up-regulate the expression of Snail and LXR,increase the secretion of IL-1β in coculture system which were consistent with 25HC.